Transforming growth factor beta, or "TGF-.beta." as used hereafter, refers to a family of multifunctional, dimeric polypeptides having a molecular weight of about 25000 daltons. See U.S. Pat. No. 4,931,548 to Lucas et al., the disclosure of which is incorporated by reference, as well as Lyons et al., Eur. J. Biochem 187: 467-473 (1990); Massague, Ann. Rev. Cell Biol. 6: 597-641 (1990); Roberts et al., in Peptide Growth Factors And Their Receptors, part 1 (Sporn et al., ed), pp. 419-472 (1990); Sporn et al., Science 233: 532-534 (1986); Massague, Trends in Biochem. Sci. 10: 239-240 (1985). The TGF-.uparw. have been found to stimulate certain cell types and to inhibit others with respect to cell growth and differentiation. They also influence adipogenesis, myogenesis, chondrogenesis, osteogenesis, epithelial cell differentiation and immune cell function. See Lucas et al., supra.
At least three related isoforms of TGF-.beta. have been identified, i.e., "TGF-.beta.1, TGF-.beta.2 and TGF-.beta.3". Although related, their properties are not identical, as summarized by, e.g., Graycar et al., Mol. Endrocrinol 3: 1977-1986 (1989), Cheifetz et al., J. Biol. Chem. 265:20533-10538 (1990). Promoter regions of the three isoforms vary considerably, and their production is differently regulated, as pointed out by Roberts et al., Ciba Found. Symp. 157:7-28 (1991).
TGF-.beta. molecules have been observed to be produced in an inactive, high molecular weight forms. For example, TGF-.beta.1, isolated from human and rat platelets, have been found as a complex of three components, referred to hereafter as the "large latent complex", the "LL" complex or "LLTGF-.beta.1". This complex consists of a dimer of the active TGF-.beta.1 molecule, i.e., the 25 KDa structure referred to supra. It also includes a molecular moiety referred to as the "latency associated peptide" or ".beta.1-LAP", and a larger molecule, referred to as the latent TGF-.beta.1 binding protein or "LTBP". As to the high molecular weight forms, see Pircher et al., Canc. Res. 44: 5538-5543 (1984); Wakefield et al., J. Cell Biol. 105:965-975 (1987). As to .beta.1-LAP and LTBP, see Miyazono et al., J. Biol. Chem. 263: 6407-6415 (1988); Wakefield et al., J. Biol. Chem. 263: 7646-7654 (1988); and Okada et al., J. Biochem 106: 304-310 (1989). Latent TGF-.beta.1 can be activated in vitro via various physical and chemical treatments or by exposure to low or high pH (Brown et al., Growth Factors 3: 35-43 (1990)). The activating mechanism in vivo remains unclear, but may involve enzymatic digestion, as suggested by Miyazono et al., Ciba Found. Symp. 57:81-89 (1991).
The three recognized forms of TGF-.beta. have been produced, in recombinant form, where each form of TGF-.beta. dimer is non covalently associated with the .beta.-LAP. These complexes are inactive, have a molecular mass of about 100 KDa, and are activated to produce the mature and active TGF-.beta. dimer. See Brown, supra; Gentry et al., Mol. Cell Biol. 7:3418-3427 (1987). The complex of TGF-.beta. and .beta.-LAP is referred to as a "small, latent TGF-.beta. complex".
The role of LTBP in vivo is not completely clear. It has been found to be involved in the manufacture and secretion of TGF-.beta.1 by a human erythroleukemia cell line. (Miyazono et al., EMBO J 10: 1091-1101 (1991)). The cDNA for the molecule has been cloned, and the protein contains several epidermal growth factor like repeats. See Kanzaki et al., Cell 61:1051-1061 (1990); Tsuji et al., Proc. Natl. Acad. Sci. USA 87:8835-8839 (1990). This feature is shared with many other molecules. An additional repeating structure is also found, which has 8 cysteine residues in one motif.
The structure of the LL-TGF.beta.1 complex has been analyzed in some detail, and is as described supra; however, the LL complexes of TGF-.beta.2 and TGF-.beta.3 have not been studied. Given the fact that the TGF-.beta.2 and TGF-.beta.3 molecules differ from TGF-.beta.5, and that their associated "LAP" proteins differ, it would have been expected that there would be a binding protein specific to each form and differing from that associated with TGF-.beta.1. Surprisingly, it has been found that the binding protein for both TGF-.beta.2 and TGF-.beta.3 is the same as that for TGF-.beta.1. Isolated large latent complexes are thus described which contain (i) either dimerized TGF-.beta.2 or TGF-.beta.3, (ii) the B-LAP for the TGF-.beta.2 or TGF-.beta.3 form, and (iii) the LTBP molecule, which was previously associated only with TGF-.beta.1. The complexes are useful as inactive forms of TGF-.beta.2 and TGF-.beta.3, which can be treated to yield the active TGF-.beta.2 and TGF-.beta.3 molecules.
The investigations described herein led to a surprising discovery in that an additional binding protein immunologically distinct from LTBP and having a molecular mass of about 150 KDa associates with all of TGF-.beta.1, TGF-.beta.2 and TGF-.beta.3. This is referred to as "LTBP-2" hereafter. Thus, new complexes containing TGF-.beta.1 are described, as well as a second form of isolated LL-TGF-.beta.2 and LL-TGF-.beta.3 complexes. All of the complexes described herein are characterized in preferred embodiments by a molecular weight of about 210 KDa as determined by SDS-Page.
These and other aspects of the invention are elaborated upon in the disclosure which follows.